The three-dimensional (3D) structures of protein-ligand complexes can be determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Sometimes the protein's 3D structure is known in advance. This circumstance reduces the problem to a matter of determining the binding mode, also referred to as the “pose,” of the ligand. A primary method used for structure determination by NMR relies on intermolecular Nuclear Overhauser Effect (NOE) distance restraints between the protein and the ligand. These restraints can be derived from Nuclear Overhauser Effect Spectroscopy (NOESY) NMR experiments, for example, from 3D 13C-edited, 15N/13C-filtered HSQC-NOESY experiments, or from 2D 1H-1H NOESY experiments. In favorable situations, high-resolution binding modes can be determined by this method.
Various methods have been proposed for the structural study of protein-ligand complexes, some of which do not require that protein resonance assignments be made. For example, Hajduk et al. (Hajduk et al. (2004) J. Am. Chem. Soc. 126:2390-2398) describe a method purportedly useful for determining the structures of protein-ligand complexes that does not require protein resonance assignments. However, the method of Hajduk et al. is applicable only to weakly binding, soluble ligands, and does not allow for inclusion of protein resonance assignments if they are available, and therefore does not facilitate an iterative refinement process.
Similarly, Meiler & Baker describe a method purportedly useful for identifying a good fit between a set of proposed protein structures and unassigned chemical shifts, NOEs and residual dipolar couplings (Meiler & Baker, (2003) Proc. Natl. Acad. Sci. USA 100:15404-15409). The method of Meiler & Baker, however, focuses on protein structure determination and employs a Monte Carlo approach, which is may not generate the optimal results.
Dobrodumov & Gronenborn describe a method purportedly useful for identifying models of protein-protein complexes that give the best match to chemical shift changes and residual dipolar couplings (Dobrodumov & Gronenborn, (2003) Proteins 52:18-32). A drawback of this method is that the method requires protein backbone atom NMR assignments, which are sometimes not available. This method is not applicable to protein-ligand (e.g., small molecule) structures.
Xu et al., (Xu et al., (2002) Comput. Sci. Eng.4:50-62), Hus et al., (Hus et al., (2002) J. Mag. Res. 157:119-123), and Langemead, & Donald, (Langmead & Donald, (2004) J. Biomol. NMR 29:111-138) apparently describe the application of bipartite matching to the problem of assigning protein backbone resonances by matching experimental and predicted NMR data, however these reports do not address the problem of protein-ligand structure determination.
A 3D 13C-edited, 15N/13C-filtered HSQC-NOESY experimental data set contains exclusively NOE peaks between ligand protons (F3 dimension) and protein 1H13C groups (F1/F2 dimensions). A 2D 1H-1H NOESY spectrum may contain intra-ligand, protein-ligand and protein-protein NOE peaks; these can be distinguished by suitable isotopic labeling schemes. Accordingly, it is recognized that other types of NOE (e.g., 2D 1H-1H NOESY) data can readily be incorporated into the procedures described herein.
In order to derive the NOE distance restraints from 3D 13C-edited, 15N/13C-filtered HSQC-NOESY data, the ligand 1H resonances and the protein 1H,13C resonances must be assigned. The protein resonances must be re-assigned for each new ligand if a series of ligands are to be structurally characterized. While the data for assigning the bound or exchanging ligand can be collected and analyzed in a matter of days, it can take weeks or more to collect and analyze the protein assignment data. In some cases, it is difficult or even impossible to assign the protein resonances. In order to assign the intermolecular NOEs, protein backbone and side-chain assignment data sets must first be collected, processed and analyzed. If the protein assignment step could be bypassed, the utility of NMR for characterizing ligand binding modes would be greatly increased.
Thus what is needed is a high-throughput method of NMR-based structure determination of protein-ligand complexes that does not require protein resonance assignments. The present invention solves this and other problems.